T-DNA插入事件在水稻基因組的分佈特性

The Distributional Characters of T-DNA Integration Events in Rice Genome

林錦源(Jin-Yuan Lin)

指導教授 ： 范宗宸

亞洲大學/健康學院/生物科技學系碩士班/碩士(2009年)

摘要

本研究利用中央研究院余淑美博士提供的T-DNA插入致突變水稻為研究材料，先以HA-PCR進行樣品DNA的擴增，然後交由中研院基因體研究中心進行目標DNA序列的定序，再以「水稻T-DNA插入突變體兩翼序列自動化比對系統」進行兩翼序列標誌 (flanking sequence tags, FSTs) 的裁切工作，最後利用 “The Rice Annotation Project Database” 資料庫進行兩翼序列標誌之插入事件的比對分析工作。本論文總共調查1403個品系，結果獲得845個合格的兩翼序列，解序成功率約為60.23%。T-DNA插入在水稻基因組呈現非隨機性的分佈，長染色體有較高的插入機率，其中以1號染色體的機率最高(15.50%)，10號染色體的機率最低(4.02%)。但是，T-DNA插入點是否位於基因內區域 (genic regions) 與其插入點上下游各10kb距離內的基因密度並沒有顯著相關性，也與插入之染色體的長度及其20kb範圍內的基因密度是否大於平均值無顯著相關性 (卡方獨立性檢定，p>0.05) 。當T-DNA插入水稻基因OsAmy1B (Os02t0765600-01) 的外顯子 (exon) 位置時，會導致突變品系的T1種子出現穀粒褐化，以及較低的充實率與十粒重，推論此基因可能會影響穀粒發育而使充實率下降。本研究對於水稻基因組之T-DNA插入事件的分佈特性有詳實的描述，亦發現基因OsAmy1B可能會影響水稻種子發育，期待這些發現能夠供後續相關研究參考，並針對T-DNA插入位置 (insertional loci) 如何影響水稻基因表現及如何修飾水稻突變體的性狀進行更深入的探討。

關鍵字

轉移DNA(T-DNA) ； 兩翼序列標誌 ； T-DNA插入事件

並列摘要

The mutant subjects that received from Dr. Yu of Academic Sinica rendered as materials to analyze the distribution characters of T-DNA integration events in rice genome. The processes of this study were as followed: at first, we amplified the target DNA sequences by hooker adaptor PCR (HA-PCR) method, and then sequenced the PCR products by Genome Research Center, Academia Sinica. Based on the analysis data of flanking sequence tags (FSTs) alignment system, “T-DNA insert mutant flanking sequence automation BLAST system” were established. We mapped these target FSTs in a public rice database: The Rice Annotation Project Database, RAP-DB. Of all, 845 qualified FSTs in this study with a 60.23% of successful rate from 1,403 mutants. The meta-analysis demonstrated that the T-DNA integration events in the whole rice genome were non-random distributions. These events preferred large rice chromosomes to short rice chromosomes. The results indicated that highest probability (15.50%) within larger chromosome 1, and lowest (4.02%) within shorter chromosome 10. However, these events whether occurred in genic regions were unrelated the different gene densities that 10kb intervals upstream and downstream of T-DNA insertional sites. The gene densities per 20kb intervals and predicted sizes of each inserted chromosome were no relationship alike. (by the independence of chi-squared test, p>0.05). While the T-DNA inserted in the exon regions of the gene OsAmy1B (Os02t0765600-01), the mutant grain husks presented whole or partial brown colors, lower grain-fill rate and 10-grain weight. It seemed to indicate that this unexpressive gene may impact the grain developments and reduce the rate of grain-fill. In conclusion, our research rendered fine descriptions of T-DNA integration events in rice genome and we also found that the gene OsAmy1B might influence the grain developments of rice. We hope that our experiences can provide valuable data to other researchers to explore the effects of T-DNA integration events in gene expressions and to answer us those events how to modify the phenotypes of rice mutants.

並列關鍵字

transfer DNA (T-DNA) ； FSTs ； T-DNA integration events

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